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Trp Operon Regulation in E.coli
Introduction: Regulation of the trp operon gene expression DNA of single-cell prokaryotic organism (e.g. Escherichia coli ), contain approximately 4.0 millions of nucleotide pairs that form about 4300 protein-coding genes. Most of these genes are regulated and expressed according to environmental conditions. Francois Jacob and Jacques Monod put the idea that genes are switched off and on, and how it is carried out forward in their operon mode. They proposed that the regulation of protein synthesis is achieved at the genetic level and is carried by regulatory proteins called repressors (Lodish H, Berk A, Zipursky SL, et al., 2000). In E.coli, the trp operon consists of the repressor, promoter, operator and the structural genes (The trpE, trpD, trpC, trpB, and trpA). The repressor proteins actively switch off genes; it regulate expression by binding to a DNA sequence, called the operator. Repressor activity is sensitive to a ligand that binds to the repressor and signals the environmental conditions, such as nutrient levels, which provides a mechanism by which bacteria can adjust their metabolism accordingly. In E.coli, trp operon as a classical example, encodes enzymes that are needed for the biosynthesis of an essential amino acid tryptophan for survival. The trp is considered a repressible system, because the presence of tryptophan represses the expression of these genes. The trpE, trpD, trpC, trpB, and trpA genes encode enzymes involved in tryptophan biosynthesis. Tryptophan is sufficiently present in their growth environment; E.coli very sensibly cease or control biosynthesis of tryptophan in the cell. The trpR and trpL genes are involved in regulating the trp operon using two different mechanisms- trpR gene encodes the trp repressor protein, and trpL mediate the regulation of the trp operon by a mechanism called attenuation. Function of trp operon Tryptophan is one of the aromatic amino acids used in the cells, as an important constituent of proteins. When the concentration of tryptophan is low in E.coli, genes that encode enzymes for tryptophan production becomes expressed in the trp operon. These enzymes participate in metabolic pathway in which the end product is tryptophan. The overall function of the trp operon is to ensure that E.coli cell has a supply of tryptophan at all times. Mechanisms of trp regulation The operon can be regulated using two either of the two mechanisms: trp repressor protein and attenuation. The trp operon is regulated by the trp operon that binds to the trp operator, when tryptophan, a corepressor is present. A second mechanism for trp operon regulation is through attenuation, in which the formation of a terminator stem-loop causes early termination of transcription. Trp operon consist of regulatory or control region and structural genes (trpE, trpD, trpC, trpB, and trpA). TrpR- repressor regulates the operon, P''/O- Promoter; operator sequence is found in the promoter, RNA polymerase binding site, ''trp L-'' Leader sequence; attenuator (A) sequence is found in the leader, ''trp E-'' Gene for anthranilate synthetase subunit, ''trp D'' - Gene for anthranilate synthetase subunit, ''trp C'' - Gene for glycerolphosphate synthetase, ''trp B-'' Gene for tryptophan synthetase subunit, and ''trp A- ''Gene for tryptophan synthetase subunit (Brooker R. 2012) ''P/O-Promoter; operator sequence is found in the promoter, RNA polymerase binding site, trp L-'' Leader sequence; attenuator (A) sequence is found in the leader, ''trp E-'' Gene for anthranilate synthetase subunit, ''trp D'' - Gene for anthranilate synthetase subunit, ''trp C'' - Gene for glycerolphosphate synthetase, ''trp B-'' Gene for tryptophan synthetase subunit, and ''trp A-'' Gene for tryptophan synthetase subunit. Trp Operon Repressor Protein At low low tryptophan levels , tryptophan does not bind to the trp repressor protein, which prevent the repressor protein from binding to the operator site. the inability of tryptophan to bind to trp repressor protein, facilitate the binding of RNA polymerase to the operator, thus RNA polymerase can transcribe the operon, which leads to the expression of the trpE, trpD, trpC, trpB, and trpA. These genes encode enzymes involved in tryptophan biosynthesis. When tryptophan levels are high, tryptophan acts as a corepressor that binds to the trp repressor protein. The tryptophan-trp repressor complex then binds to the operator site to inhibit transcription. '''Attenuation of Trp Operon' Attenuation is a second method of trp operon regulation, which occurs when tryptophan levels are sufficient for protein synthesis. At low Tryptophan levels: Coupled transcription and translation occur when the concentration of tryptophan is very low. The ribosome pauses at the trp codons in the trpL gene, because of insufficient amounts of charged tRNATrp are present. This pause blocks the region 1 of the mRNA, so region 2 can hydrogen bond only with region 3 ( 2-3 stem-loop forms). When this happens, the 3-4 stem-loop structure cannot form. Transcriptional termination does not occur, and RNA polymerase transcribes the rest of the operon At high Tryptophan levels: coupled transcription and translation occur the concentration of tryptophan is in sufficient amount or high in the cell. Translation of the trpL gene progresses to its stop codon, where the ribosome pauses. This blocks region 2 from hydrogen bonding with any region and thereby enables region 3 to hydrogen bond with region 4 (3-4 stem-loop forms). This terminates transcription at the U-rich attenuator stimulus for trp gene expression Low tryptophan level stimulates the transcription of the entire trp operon. At high tryptophan levels, the trp operon is repressed, by repressor protein or by attenuation, thus the trp gene is not expression or activated Why trp gene expression is regulated Trp operon/genes are transcriptionally repressible, since cell generally uses only a fraction of its genome at any given moment in time, when required, genes could be switched on, or expressed, but only for as long as needed. In this way, the cell could avoid wasteful production of unnecessary transcripts and proteins. In other words trp gene expression is regulated, so that tryptophan is produced only when it is required; thus cells avoid wasting valuable energy in transcription of tryptophan when is not needed. It enables E.coli to compete as efficient as possible for the limited resources, that enable their survival (natural selection). Also, trp gene is regulated because bacteria exist in an environment that is constantly changing with regard to temperature, nutrients, and many other factors (Brooker R. 2012). References Brooker, R. (2012). 14.2. In Genetics: Analysis and principles. (4th ed., pp. 371-375). S.l.: Mcgraw Hill Higher Educat. Lodish, H., Berk, A., Zipursky, S., Matsudaira, P., Baltimore, D., & Darnell, J. (2000). 10.1. In Molecular cell biology (4th ed.). New York: W.H. Freeman Simao, E., Remy, E., Thieffry, D., & Chaouiya, C. (2005). Qualitative modelling of regulated metabolic pathways: Application to the tryptophan biosynthesis in E.Coli. Bioinformatics, Ii190-Ii196. Tabaka, M., Cybulski, O., & Hołyst, R. (2008). Accurate Genetic Switch in Escherichia coli: Novel Mechanism of Regulation by Co-repressor. Journal of Molecular Biology, 1002-1014.